a) Field of the Invention
The present invention relates generally to microdevices and methods of using those devices for the parallel, in vitro screening of a plurality of biomolecule-analyte interactions. More specifically, the present invention relates to use of the devices for drug development, functional proteomics, and clinical diagnostics.
b) Description of Related Art
A vast number of new drug targets are now being identified using a combination of genomics, bioinformatics, genetics, and high-throughput biochemistry. Genomics provides information on the genetic composition and the activity of an organism""s genes. Bioinformatics uses computer algorithms to recognize and predict structural patterns in DNA and proteins, defining families of related genes and proteins. The information gained from the combination of these approaches is expected to greatly boost the number of drug targets (usually, proteins).
The number of chemical compounds available for screening as potential drugs is also growing dramatically due to recent advances in combinatorial chemistry, the production of large numbers of organic compounds through rapid parallel and automated synthesis. The compounds produced in the combinatorial libraries being generated will far outnumber those compounds being prepared by traditional, manual means, natural product extracts, or those in the historical compound files of large pharmaceutical companies.
Both the rapid increase of new drug targets and the availability of vast libraries of chemical compounds creates an enormous demand for new technologies which improve the screening process. Current technological approaches which attempt to address this need include multiwell-plate based screening systems, cell-based screening systems, microfluidics-based screening systems, and screening of soluble targets against solid-phase synthesized drug components.
Automated multiwell formats are the best developed high-throughput screening systems. Automated 96-well plate-based screening systems are the most widely used. The current trend in plate based screening systems is to reduce the volume of the reaction wells further, thereby increasing the density of the wells per plate (96-well to 384- and 1536-well per plate). The reduction in reaction volumes results in increased throughput, dramatically decreased bioreagent costs, and a decrease in the number of plates which need to be managed by automation.
However, although increases in well numbers per plate are desirable for high throughput efficiency, the use of volumes smaller than 1 microliter in the well format generates significant problems with evaporation, dispensing times, protein inactivation, and assay adaptation. Proteins are very sensitive to the physical and chemical properties of the reaction chamber surfaces. Proteins are prone to denaturation at the liquid/solid and liquid/air interfaces. Miniaturization of assays to volumes smaller than 1 microliter increases the surface to volume ratio substantially. (Changing volumes from 1 microliter to 10 nanoliter increases the surface ratio by 460%, leading to increased protein inactivation.) Furthermore, solutions of submicroliter volumes evaporate rapidly, within seconds to a few minutes, when in contact with air. Maintaining microscopic volumes in open systems is therefore very difficult.
Other types of high-throughput assays, such as miniaturized cell-based assays are also being developed. Miniaturized cell-based assays have the potential to generate screening data of superior quality and accuracy, due to their in vivo nature. However, the interaction of drug compounds with proteins other than the desired targets is a serious problem related to this approach which leads to a high rate of false positive results.
Microfluidics-based screening systems that measure in vitro reactions in solution make use of ten to several-hundred micrometer wide channels. Micropumps, electroosmotic flow, integrated valves and mixing devices control liquid movement through the channel network. Microfluidic networks prevent evaporation but, due to the large surface to volume ratio, result in significant protein inactivation. The successful use of microfluidic networks in biomolecule screening remains to be shown.
Drug screening of soluble targets against solid-phase synthesized drug components is intrinsically limited. The surfaces required for solid state organic synthesis are chemically diverse and often cause the inactivation or non-specific binding of proteins, leading to a high rate of false-positive results. Furthermore, the chemical diversity of drug compounds is limited by the combinatorial synthesis approach that is used to generate the compounds at the interface. Another major disadvantage of this approach stems from the limited accessibility of the binding site of the soluble target protein to the immobilized drug candidates.
Miniaturized DNA chip technologies have been developed (for example, see U.S. Pat. Nos. 5,412,087, 5,445, 934 and 5,744,305) and are currently being exploited for nucleic acid hybridization assays. However, DNA biochip technology is not transferable to protein assays because the chemistries and materials used for DNA biochips are not readily transferable to use with proteins. Nucleic acids withstand temperatures up to 100xc2x0 C., can be dried and re-hydrated without loss of activity, and can be bound directly to organic adhesion layers supported by materials such as glass while maintaining their activity. In contrast, proteins must remain hydrated, kept at ambient temperatures, and are very sensitive to the physical and chemical properties, of the support materials. Therefore, maintaining protein activity at the liquid-solid interface requires entirely different immobilization strategies than those used for nucleic acids. Additionally, the proper orientation of the protein at the interface is desirable to ensure accessibility of their active sites with interacting molecules. With miniaturization of the chip and decreased feature sizes the ratio of accessible to non-accessible antibodies becomes increasingly relevant.
In addition to the goal of achieving high-throughput screening of compounds against targets to identify potential drug leads, researchers also need to be able to identify highly specific lead compounds early in the drug discovery process. Analyzing a multitude of members of a protein family or forms of a polymorphic protein in parallel (multitarget screening) enables quick identification of highly specific lead compounds. Proteins within a structural family share similar binding sites and catalytic mechanisms. Often, a compound that effectively interferes with the activity of one family member also interferes with other members of the same family. Using standard technology to discover such additional interactions requires a tremendous effort in time and costs and as a consequence is simply not done.
However, cross-reactivity of a drug with related proteins can be the cause of low efficacy or even side effects in patients. For instance, AZT, a major treatment for AIDS, blocks not only viral polymerases, but also human polymerases, causing deleterious side effects. Cross-reactivity with closely related proteins is also a problem with nonsteroidal anti-inflammatory drugs (NSAIDs) and aspirin. These drugs inhibit cyclooxygenase-2, an enzyme which promotes pain and inflammation. However, the same drugs also strongly inhibit a related enzyme, cyclooxygenase-1, that is responsible for keeping the stomach lining and kidneys healthy, leading to common side-effects including stomach irritation.
The miniaturized, parallel screening of a plurality of protein interactions is also useful and necessary for a number of applications beyond high-throughput drug screening. For instance, the function of newly discovered proteins could be assayed effectively in a parallel format with a plurality of potential ligands or potential substrates of known protein families. Also, miniaturized diagnostic devices which allow for the analysis of a plurality of analytes by binding the analytes to proteins such as antibodies would be desirable.
For the foregoing reasons, there is a need for a miniaturized device and methods of using the device for the parallel, in vitro, screening of a plurality of biomolecular interactions, especially the interactions of proteins with analytes or other proteins.
The present invention is directed to a device and methods of use of the device that satisfy the need for the parallel, in vitro, screening of a plurality of biomolecular interactions, especially the interactions of proteins with analytes or other proteins.
One embodiment of the present invention provides a device for analyzing components of a fluid sample, comprising a plurality of noncontiguous reactive sites. Each of the reactive sites comprises a substrate, an organic thinfilm chemisorbed or physisorbed on a portion of a surface of the substrate, and a biological moiety immobilized on the organic thinfilm, wherein each of the reactive sites may independently react with a component of the fluid sample and are separated from each other by a region of the substrate that is free of organic thinfilm
In a particularly preferred embodiment of the device, each of the reactive sites on the device of the invention is in a microchannel oriented parallel to microchannels of other reactive sites on the device, where the microchannels are microfabricated into or onto the substrate.
An alternative embodiment of the invention provides a device for analyzing components of a fluid sample that comprises a substrate, a plurality of parallel microchannels microfabricated into or onto said substrate, and a biological moiety immobilized within at least one of the parallel microchannels in such a way that the biological moiety may interact with a component of the fluid sample. In a preferred embodiment, the biological moiety is a protein.
Methods of using the devices of the invention are also provided by the present invention. In one embodiment, the invention provides for a method of screening a plurality of biological moieties in parallel for their ability to interact with a component of a fluid sample. This method comprises first delivering the fluid sample to the reactive sites of the invention device, where each of the different biological moieties is immobilized on a different reactive site of the device and detecting, either directly or indirectly, for the interaction of the component with the immobilized biological moiety at each reactive site. The interaction being assayed may be a binding interaction, catalysis, or translocation by a membrane protein through a lipid bilayer.
In an alternative embodiment of the invention, the device of the invention is used to screen a plurality of components, each in separate fluid samples, for their ability to interact with a biological moiety. The method of this embodiment comprises first delivering each of the different fluid samples to separate reactive sites of the invention device, wherein the separate reactive sites of the device each comprise the immobilized biological moiety. The next step comprises detecting, either directly or indirectly, for the interaction of the immobilized biological moiety at each reactive site with the component delivered to that reactive site. Again, the interaction being assayed may be a binding interaction, catalysis, or translocation by a membrane protein through a lipid bilayer.
In another embodiment of the present invention, a similar method is used to screen a fluid sample for the presence or amount of a plurality of analytes (in parallel). This method has potential applications in diagnostics. The method comprises delivering the fluid sample to a plurality of reactive sites on the invention device, wherein each of the reactive sites comprises an immobilized biological moiety which can either react, bind, or otherwise interact with at one of said plurality of analytes. The method also comprises a final step of detecting for the interaction of the analyte with the immobilized biological moiety of each reactive site.
In another embodiment of the invention, the device may also be used to screen a plurality of binding candidates in parallel for their ability to bind to a biological moiety. In the method of this embodiment, different fluid samples, each containing a different binding candidate (or a different mixture of binding candidates) to be tested, are delivered separate reactive sites of the invention device, wherein the separate reactive sites each comprise the immobilized biological moiety. The next step of the method comprises detecting, either directly or indirectly, for the presence or amount of the binding candidate.
The present invention also provides for methods of determining in parallel whether or not each of a plurality of proteins belong to a certain protein family based on either binding to a common ligand or reactivity with a common substrate. These methods involve delivering a fluid sample comprising a ligand or substrate of a known protein family to the reactive sites of the invention device which each contain one of the different proteins to be assayed and then detecting, either directly or indirectly, for binding or reaction with the known ligand that is characteristic of the protein family.